Posttranslation modification of proteins is complex and highly regulated with importance for many cellular processes. The reversible modification of lysine residues by acetylation of the ε-amino group has attracted much attention recently (Cohen and Yao, Science STKE, 2004). Initially, the reversible acetylation of N-terminal lysine residues in histone proteins was described (Marks et al., Nat. Rev. Cancer 1, 194-202, 2001). Histone proteins H2A/B, H3 and H4 are forming the octameric histone core complex of chromatin. The complex N-terminal modifications at lysine residues by acetylation or methylation and at serine residues by phosphorylation constitute part of the so called “histone code” (Stahl & Ellis, Nature 403, 41-45, 2000). In a simple model, acetylation of positively charged lysine residues decreases affinity to negatively charged DNA, which now becomes accessible for the entry of transcription factors. Thus, reversible modification of lysine residues within core histone proteins was understood as being important for gene regulation. Histone acetylation and deacetylation is catalysed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. For example, the HDAC isoenzymes HDAC1 or 2 are associated with transcriptional repressor complexes, switching chromatin to a transcriptionally inactive, silent structure (Marks et al. Nature Cancer Rev 1, 194-202, 2001). The opposite holds true for certain HATs which are associated with transcriptional activator complexes.
Three different classes of HDACs have been described so far, namely class I (HDAC 1-3, 8) with Mr=42-55 and class II (HDAC 4-7, 9, 10) with Mr=120-130 kDa, both sensitive towards inhibition by Trichostatin A (TSA). The class III (Sir2 homologues, SIRTs) enzymes which are quite distinct by their NAD+ dependency and TSA insensitivity (Ruijter et al. Biochem. J. 370, 737-749, 2003; Khochbin et al. Curr. Opin. Gen. Dev. 11, 162-166, 2001; Verdin et al. Trends Gen 19, 286-293, 2003). HDAC 11 with Mr=39 kDa was cloned recently and displayed homology to class I and II family members (Gao et al. J. Biol. Chem. 277, 25748-25755, 2002). Those HATs and HDACs important for transcriptional regulation exist in large complexes together with transcription factor and platform proteins in cells (Fischle et al. Mol. Cell 9, 45-47, 2002). Surprisingly, only about 2% of all genes are regulated by histone acetylation as estimated based on differential display analysis of 340 genes and TSA as the reference HDI (von Lint et al. Gene Expression 5, 245-253, 1996). New studies with the HDAC inhibitor SAHA in multiple myeloma cells showed that these transcriptional changes can be grouped into distinct functional gene classes important for e.g. regulation of apoptosis or proliferation (Mitsiades et al. Proc. Natl. Acad. Sci. 101, pp 540, 2004).
As said before, there is growing evidence for substrates of HDACs different to histone proteins and regulation of processes different to gene transcription (Johnstone & Licht, Cancer Cell 4, 13-18, 2003, Cohen and Yao, Science STKE, 2004). Thus, the correct name for HDACs should be lysine-specific protein deacetylases. As a consequence of these findings, inhibitors of HDACs should not only effect chromatin structure and gene regulation but also protein function and stability by regulating protein acetylation in general. HDAC6 was identified independently in 1999 by Grozinger et al. (Proc. Natl. Acad. Sci. 96, 4868-4873) and Verdel et al. (J. Biol. Chem. 274, 2440-2448) as a HDAC class II enzyme with substrates different to core histone proteins. With 1216 amino acids, HDAC6 is the largest HDAC isoenzyme yet identified in humans and unique with HDAC10 by having two internal ε-acetyllysine specific deacetylation domains, both important for enzymatic activity. HDAC6 is mainly expressed in the cytoplasm and co-localizes with microtubule structures. Microtubles are dynamic structures formed by α/β-tubulin heterodimers, polymerizing parallel to a cylindrical axis. It has long been known that tubulin is modified on lysine residues by acetylation, e.g. lysine residue 40 on α-tubulin, thereby stabilizing tubulin structure and dynamics. HDAC6 was identified as a protein binding to αβ-tubulin, deacetylating α-tubulin and antagonizing the tubulin hyperacetylation induced by tubulin stabilizing anti-cancer agents like taxol (Zhang et al. EMBO J. 22, 1168-1179, 2003; Hubbert et al. Nature 417, 455-458, 2002).
Different publications highlight the pathophysiological importance of HDAC6 in processes like cell migration, protein folding/degradation and apoptosis. In breast cancer, HDAC6 was described as an estrogen induced gene and HDAC6 overexpression enhanced cell migration (Saji et al. Oncogene 2005, 24, 4531-4539). In model experiments, inhibition of HDAC6 by the small molecule inhibitor Tubacin had comparable effect as the anti-estrogen Tamoxifen. Most importantly, Kaplan-Maier analysis of estrogen receptor (ER) positive breast cancer patients showed that those patients with ER and HDAC6 expression responded best to continuous adjuvant treatment with Tamoxifen.
It had been described that Hsp90 is regulated by acetylation and broad class I/II specific HDAC inhibitors like LBH589 induce Hsp90 hyperacetylation (George et al. Blood 105, 1768-76, 2005). The chaperone Hsp90 is well recognized as a key player in stabilization of oncoproteins like mutant raf kinase or overexpressed HER2 receptor tyrosine kinase (Maloney & Workman, Expert Opin. Biol. Ther. 2, 3-24, 2002). The Hsp90 inhibitor 17-allylamino-demethoxy-geldanamycin (17-AAG) is currently tested in clinical phase 1 studies (Ramanathan et al., Clin. Canc. Res. 11, 3385-391, 2005). Kovacs et al. now showed that the acetylated chaperone Hsp90 is a substrate of HDAC6 with deacetylated Hsp90 as the functional ATP binding enzyme in complex with the co-chaperone p23 and the glucocorticoid receptor (Mol. Cell 18, 601-607, 2005). Another function of HDAC6 in protein turnover, namely the clearance of misfolded polyubiquitinylated proteins via the aggresome, was described by Kawaguchi (Cell 115, 727-738, 2003). This interaction is mediated via the C-terminal polyubiquitin associated zinc finger (PAX) domain (Hook et al. Proc. Natl. Acad. Sci. 99, 13425-430, 2002).
Finally, HDAC6 was discussed as a target for chemosensitization towards stabilizing tubulin inhibitors, namely paclitaxel and docetaxel (Marcus et al. Cancer Res. 65, 3883-3893, 2005). This synergism was most pronounced by combination of paclitaxel with the farnesyltransferase inhibitor Sarasar (SCH66336, Ionafarnib). In summary, it is highly likely that, by selective inhibition of HDAC6, various pathological conditions can be treated, in particular cancer.
HDAC inhibitors from various chemical classes were described in the literature with four most important classes, namely (i) hydroxamic acid analogs, (ii) benzamide analogs, (iii) cyclic peptides/peptolides and (iv) fatty acid analogs. A comprehensive summary of known HDAC inhibitors was published recently by Miller et al. (J. Med. Chem. 46, 5097-5116, 2003). There is only limited data published regarding specificity of these histone deacetylase inhibitors. In general, most hydroxamate based HDAC inhibitors are not specific regarding class I and II HDAC enzymes. For example, TSA inhibits HDACs 1, 3, 4, 6 and 10 with IC50 values around 20 nM, whereas HDAC8 was inhibited with IC50=0.49 μM (Tatamiya et al, AACR Annual Meeting 2004, Abstract #2451). In addition, data on class I selectivity of benzamide HDIs are emerging. The benzamide analog MS-275, developed by Schering AG/Berlex in clinical phase I, inhibited class I HDAC1 and 3 with IC50=0.51 μM and 1.7 μM, respectively. In contrast class II HDACs 4, 6, 8 and 10 were inhibited with IC50 values of >100 μM, >100 μM, 82.5 μM and 94.7 μM, respectively (Tatamiya et al, AACR Annual Meeting 2004, Abstract #2451). A comprehensive set of pharmacological data is published on these class I or class I/II selective HDAC inhibitors. They are effective directly via induction of histone hyperacetylation on a transcriptional level, up- or down regulating cancer relevant genes. These genes include p21CIP1, Cyclin E, transforming growth factor β (TGFβ), p53 or the von Hippel-Lindau (VHL) tumor suppressor genes, which are upregulated, whereas Bcl-XL, bcl2, hypoxia inducible factor (HIF)1α, vascular endothelial growth factor (VEGF) and cyclin A/D are down-regulated by HDAC inhibition (reviewed by Kramer et al. Trends Endocrin. Metabol. 12, 294-300, 2001).
Interestingly, only very few data is published describing isotype-selective HDAC inhibitors.
The group of S. Schreiber described a hydroxamate analog named Tubacin as a selective HDAC6 inhibitor (Haggarty et al. Proc. Natl. Acad. Sci. USA 100, 4389-4394, 2003). In initial experiments Tubacin induced tubulin hyperacetylation and decreased cell migration. Therefore, a pharmacological activity of an HDAC6 selective inhibitor in treating advanced cancer patients with metastatic disease is highly likely.
There is growing rational for synergism of class I and class I/II specific HDAC inhibitors with chemotherapeutic as well as target specific cancer drugs. For example, synergism was shown for SAHA with the kinase/cdk inhibitor flavopiridol (Alemenara et al. Leukemia 16, 1331-1343, 2002), for LAQ-824 with the bcr-abl kinase inhibitor Glivec in CML cells (Nimmanapalli et al. Cancer Res. 63, 5126-5135, 2003), for SAHA and Trichostatin A (TSA) with etoposide (VP16), cisplatin and doxorubicin (Kim et al. Cancer Res. 63, 7291-7300, 2003) and LBH589 with the Hsp90 inhibitor 17-AAG (George et al. Blood 105, 1768-76, 2005). It is highly likely that a selective HDAC6 inhibitor also synergizes with established chemotherapeutic as well as targeted cancer drugs, e.g. taxanes or epothilones as tubulin stabilizing agents.
Clinical studies in cancer with class I and class I/II selective HDAC inhibitors are on-going, namely with SAHA (Merck Inc.), Valproic acid, FK228/Depsipeptide (Gloucester Pharmaceuticals/NCI), MS275 (Berlex-Schering), NVP LBH-589 (Novartis), PXD-101 (Topotarget/Curagen), MGCD0103 (Methylgene Inc), Valproic acid (G2M Cancer Drugs/Topotarget) and Pivaloyloxymethylbutyrate/Pivanex (Titan Pharmaceuticals). These studies showed first evidence of clinical efficacy, highlighted recently by partial and complete responses with FK228/Depsipeptide in patients with peripheral T-cell lymphoma (Plekarz et al. Blood, 98, 2865-2868, 2001). To our knowledge, no clinical development of an isotype-selective HDAC inhibitor has been reported so far.
Recent publications showed possible medical use of class I/II specific HDAC inhibitors in diseases different to cancer. These diseases include systemic lupus erythematosus (Mishra et a. J. Clin. Invest. 111, 539-552, 2003; Reilly et al. J. Immunol. 173, 4171-4178, 2004), rheumatoid arthritis (Chung et al. Mol, Therapy 8, 707-717, 2003; Nishida et al. Arthritis & Rheumatology 50, 3365-3376, 2004), inflammatory diseases (Leoni et al. Proc. Natl. Acad. Sci. USA 99, 2995-3000, 2002) and neurodegenerative diseases like Huntington's disease (Steffan et al. Nature 413, 739-743, 2001, Hockly et al. Proc. Natl. Acad. Sci. USA 100(4):2041-6, 2003). It is likely that isotype selective inhibitors are also pharmacologically active in these diseases. As such, HDAC6 has been described as a factor in the organization of the T-cell receptor/antigen presenting cell immune synapse (Serrador et al. Immunity 20, 417-428, 2004).
Cancer chemotherapy was established based on the concept that cancer cells with uncontrolled proliferation and a high proportion of cells in mitosis are killed preferentially. Standard cancer chemotherapeutic drugs finally kill cancer cells upon induction of programmed cell death (“apoptosis”) by targeting basic cellular processes and molecules, namely RNA/DNA (alkylating and carbamylating agents, platin analogs and topoisomerase inhibitors), metabolism (drugs of this class are named anti-metabolites) as well as the mitotic spindle apparatus (stabilizing and destabilizing tubulin inhibitors). Class I and class I/II selective inhibitors of histone deacetylases constitute a new class of anti cancer drugs with differentiation and apoptosis inducing activity. It is highly likely that isotype selective inhibitors have a defined activity profile and a broad therapeutic index. In this regard HDAC6 selective inhibitors might be active in cancer therapy by, for example, inhibiting cell migration, synergizing with agents targeting the mitotic spindle or effecting dysregulated protein folding and degradation via the chaperone and proteasome/aggresome machineries.